Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naïve ...cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naïve cell, resulting in productive viral replication in the naïve cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures.
The mechanisms by which enteric commensal microbiota influence maturation and repair of the epithelial barrier are relatively unknown. Epithelial restitution requires active cell migration, a process ...dependent on dynamic turnover of focal cell-matrix adhesions (FAs). Here, we demonstrate that natural, commensal bacteria stimulate generation of reactive oxygen species (ROS) in intestinal epithelia. Bacteria-mediated ROS generation induces oxidation of target cysteines in the redox-sensitive tyrosine phosphatases, LMW-PTP and SHP-2, which in turn results in increased phosphorylation of focal adhesion kinase (FAK), a key protein regulating the turnover of FAs. Accordingly, phosphorylation of FAK substrate proteins, focal adhesion formation, and cell migration are all significantly enhanced by bacterial contact in both in vitro and in vivo models of wound closure. These results suggest that commensal bacteria regulate cell migration via induced generation of ROS in epithelial cells.
Surface plasmon resonance is a well-established technology for real-time highly sensitive label-free detection and measurement of binding kinetics between biological samples. A common drawback, ...however, of surface plasmon resonance detection is the necessity for far field angular resolved measurement of specular reflection, which increases the size as well as requiring precise calibration of the optical apparatus. Here we present an alternative optoelectronic approach in which the plasmonic sensor is integrated within a photovoltaic cell. Incident light generates an electronic signal that is sensitive to the refractive index of a solution via interaction with the plasmon. The photogenerated current is enhanced due to the coupling of the plasmon mode with Fabry-Pérot modes in the absorbing layer of the photovoltaic cell. The near field electrical detection of surface plasmon resonance we demonstrate will enable a next generation of cheap, compact and high throughput biosensors.
Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple ...sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38-100% and 75.76-96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We utilized our in situ method for the one-step assembly of single-layer electrochromic devices (ECDs) with a 3,4-propylenedioxythiophene (ProDOT) acrylate derivative, and long-term stability was ...achieved. By coupling the electroactive monomer to the cross-linkable polymer matrix, preparation of the electrochromic ProDOT polymer can occur followed by UV cross-linking. Thus, we achieve immobilization of the unreacted monomer, which prevents any degradative processes from occurring at the counter electrode. This approach eliminated spot formation in the device and increased stability to over 10 000 cycles when compared to 500 cycles with conventional ProDOT devices wherein the monomer is not immobilized. The acrylated electrochromic polymer exhibits similar electrochromic properties as conventional ProDOT devices, such as photopic contrast (48% compared to 46%) and switch speed (both 2 s). This method can be applied to any one-layer electrochromic system where improved stability is desired.
This article presents the development of a stretchable sensor network with high signal-to-noise ratio and measurement accuracy for real-time distributed sensing and remote monitoring. The described ...sensor network was designed as an island-and-serpentine type network comprising a grid of sensor "islands" connected by interconnecting "serpentines." A novel high-yield manufacturing process was developed to fabricate networks on recyclable 4-inch wafers at a low cost. The resulting stretched sensor network has 17 distributed and functionalized sensing nodes with low tolerance and high resolution. The sensor network includes Piezoelectric (PZT), Strain Gauge (SG), and Resistive Temperature Detector (RTD) sensors. The design and development of a flexible frame with signal conditioning, data acquisition, and wireless data transmission electronics for the stretchable sensor network are also presented. The primary purpose of the frame subsystem is to convert sensor signals into meaningful data, which are displayed in real-time for an end-user to view and analyze. The challenges and demonstrated successes in developing this new system are demonstrated, including (a) developing separate signal conditioning circuitry and components for all three sensor types (b) enabling simultaneous sampling for PZT sensors for impact detection and (c) configuration of firmware/software for correct system operation. The network was expanded with an in-house developed automated stretch machine to expand it to cover the desired area. The released and stretched network was laminated into an aerospace composite wing with edge-mount electronics for signal conditioning, processing, power, and wireless communication.
Continuous exposure to high ambient temperatures brings about a number of oxidative damages in chickens. Copper (Cu), an active component of a number of antioxidative defence components, should ...arrest these changes to take place although that may not be possible under the standard dosing regimen followed by the industry. To ascertain the optimum dose response that may be beneficial in sustaining the performance of chickens under heat stress (HS), broiler chickens (n = 400) were exposed to high ambient temperature (between 27.2°C and 35.3°C) during 1–35 d. Copper (Cu) as Cu proteinate (Cu-P) at concentrations of 37.5, 75, 112.5, and 150 mg/kg was supplemented to the diet. The negative control (NC) diet did not contain any supplemental Cu. Increasing dietary Cu improved (P<0.001) body weight, feed intake, and conversion ratio. Serum concentrations of total cholesterol at 21 d (P=0.009), HDL cholesterol at 35 d (P=0.008), LDL cholesterol at 21 d (P=0.015), and triacylglycerol at both 21 d (P=0.033) and 35 d (P=0.001) decreased as Cu in the diet increased. As Cu in the diet increased, hemoglobin increased (P=0.003) at 21 d, and the heterophil to lymphocyte ratio decreased both at 21 d (P=0.047) and 35 d (P=0.001). Superoxide dismutase and glutathione peroxidase activities increased when dietary Cu increased to 150 mg/kg (P<0.01). Liver Cu at 35 d increased linearly with the dose of Cu in the diet (P=0.0001). Selected bacteria were enumerated in the digesta to ascertain if Cu super-dosing affected their population in any way in the absence of any enteric challenge. Escherichia coli and total Salmonella numbers decreased (P=0.0001), and total Lactobacillus increased (P=0.0001) proportionately with dietary Cu. Interleukin-6 and tumour necrosis factor-α gene expression increased linearly (P=0.0001) as Cu in the diet increased though the response plateaued at 112.5 mg/kg. It was concluded from the present experiment that during conditions of impending HS, dietary supplementation of 112.5 to 150 mg Cu/kg diet as Cu-P may be a novel strategy to alleviate the negative effects of HS without involving any apparent risk of Cu toxicity.
The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the ...method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel ...quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-gamma, MCP-1, TNF-alpha, MIP-1beta) and in human PBMC (IL-6, IL-8, TNF-alpha, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V.sup.+ PI.sup.+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical ...pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lung ENaC is redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury.
Background: Proteolytic degradation of epithelial sodium channels (ENaC) assists in regulating net salt and water balance in lung epithelia.
Results: H2O2 increases surface expression of α-ENaC, transepithelial Na transport, and alveolar fluid clearance via redox-sensitive Nedd8.
Conclusion: Redox-sensitive Nedd8 is involved in the ubiquitination of lung ENaC.
Significance: Understanding ROS-mediated signaling of lung ENaC is crucial for understanding pulmonary physiology and pathology.
PDF
